Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus

• DOI: 10.1128/jb.169.7.3168-3174.1987
• Publication Date: 2016-11-02T23:02:20Z
• Authors: R C Doten; K L Ngai; D J Mitchell; L N Ornston
• Keywords: Acinetobacter calcoaceticus; Gene cluster

The beta-ketoadipate pathway of Acinetobacter calcoaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); other catalyzed products pca protocatechuate acetyl-CoA reactions analogous or identical those sequence. We used expression plasmid pUC18 construct libraries DNA from an A. mutant strain which genes had been deleted. Immunological screening with antiserum pcaE gene product, beta-ketoadipate:succinyl-CoA transferase I, resulted in isolation a cloned 11-kilobase-pair (kbp) fragment inducibly expressed all under control lac promoter on pUC18. induced Escherichia coli cells formed at levels 10- 30-fold higher than found fully cultures, although 3,4-dioxygenase (the iron-containing product pcaA gene) recombinant possessed relatively low turnover number. An E. culture expressing quantitatively converted beta-ketoadipate; failure organism metabolize latter compound can be most readily ascribed pool succinyl-CoA, required substrate for transferase, coli. order direction transcription were determined pcACBDFE identification subclones, using natural transformation identify subclones carrying corresponding dysfunctional alleles strains, restriction mapping both 11-kbp derivatives containing Tn5 pcaA, pcaB, pcaD, genes. hybridized strongly specifically previously